Anti-pre-S1 HBV antibodies

ABSTRACT

Provided are human antibodies that specifically bind to HBV Pre-S1 domain ligand and inhibit HBV or HDV infection, antibodies binding to a set of amino acid residues that are critical for viral receptor engagement, and uses of these antibodies to prevent, or treat or diagnose HBV or HDV infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 15/864,494, filed Jan. 8, 2018, which is a continuation of U.S. application Ser. No. 15/566,555, filed Oct. 13, 2017, which is a U.S. National Phase of PCT/CN2016/082985, filed May 23, 2016, which claims priority from PCT/CN2015/079534, filed May 22, 2015, each of which is incorporated herein by reference in their entireties.

INTRODUCTION

More than one third of the world population has been infected by Hepatitis B virus (HBV), and 240 million people are presently chronically infected. HBV infection and related diseases result in about one million deaths annually.

The surface antigen of HBV is composed of Large (L), Middle (M) and Small (S) proteins. The L and M proteins have additional domains at their N terminal as compared to the S protein which only has the S domain. L contains Pre-S1, Pre-S2, and S domains; M contains Pre-S2 and S domains; S protein contains only the S domain. The pre-S1 domain in L protein is the target molecule of HBV receptor(s) expressed on human hepatic cell surface, and antibodies to the pre-S1 domain of HBV have been reported, e.g. Watashi et al, Int. J. Mol. Sci. 2014, 15, 2892-2905, refs 22-27. Relevant literature includes descriptions of the HBV receptor in WO2013159243A1, a humanized antibody from mouse hybridoma, KR127 in U.S. Pat. No. 7,115,723, and pre-S1 peptides in U.S. Pat. No. 7,892,754.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for immune-activation by inhibiting HBV and/or HDV. In one aspect, the invention provides an antibody antigen binding domain which specifically binds HBV Pre-S1, and comprises complementarity determining region (CDR) 1, CDR2 and CDR3, in a combination selected from (a)-(r) as follows, wherein the antibody (Ab), heavy chain (HC) or light chain (LC) and CDR nomenclature system (Kabat, IMGT or composite) from which the CDR combinations derive are shown in the first column, and residues in bold text are Kabat system, and residues underlined are IMGT system:

HCDRs of Unique HBV Pre-S1 Specific Antibodies

MAbs CDR1 CDR2 CDR3 m36-HC GFTFDDYA MH GT SWNSGSI GYADSVKG AKTSYGGAFDI K: SEQ ID NO: 59, res. 6-10 K: SEQ ID NO: 60 K: SEQ ID NO: 61, res. 3-11 I: SEQ ID NO: 59, res. 1-8 I: SEQ ID NO: 60, res. 2-9 I: SEQ ID NO: 61 C: SEQ ID NO: 59 C: SEQ ID NO: 60 C: SEQ ID NO: 61 m36-LC SGN TSNIGSYY AY DNN QRPS ATWDDSLNGPV K: SEQ ID NO: 62 K: SEQ ID NO: 63 K: SEQ ID NO: 64 I: SEQ ID NO: 62, res. 4-11 I: SEQ ID NO: 63, res. 1-3 I: SEQ ID NO: 64 C: SEQ ID NO: 62 C: SEQ ID NO: 63 C: SEQ ID NO: 64 71-HC GYTTGYY IH RINPNSGGTN AREGRGGMDV K: SEQ ID NO: 65, res. 5-9 K: SEQ ID NO: 66 K: SEQ ID NO: 67, res. 3-10 I: SEQ ID NO: 65, res. 1-7 I: SEQ ID NO: 66 I: SEQ ID NO: 67 C: SEQ ID NO: 65 C: SEQ ID NO: 66 C: SEQ ID NO: 67 71-LC RSS QSLLHSNGYNY LGSNRAS MQGLQPPIT K: SEQ ID NO: 68, res. 1-12  K: SEQ ID NO: 69 K: SEQ ID NO: 70 I: SEQ ID NO: 68, res. 4-14  I: SEQ ID NO: 69 I: SEQ ID NO: 70 C: SEQ ID NO: 68 C: SEQ ID NO: 69 C: SEQ ID NO: 70 76-HC GFTFSSYA MH V ISYDGSNK YYADSVKG ASGAFDI K: SEQ ID NO: 71, res. 6-10  K: SEQ ID NO: 72 K: SEQ ID NO: 73, res. 3-7 I: SEQ ID NO: 71, res. 1-8 I: SEQ ID NO: 72, res. 2-9 I: SEQ ID NO: 73 C: SEQ ID NO: 71 C: SEQ ID NO: 72 C: SEQ ID NO: 73 76-LC RSS HSLVYSDGNTYLS KVS NRDF MQGTHWPGT K: SEQ ID NO: 74 K: SEQ ID NO: 75 K: SEQ ID NO: 76 I: SEQ ID NO: 74, res. 4-14  I: SEQ ID NO: 75, res. 1-3 I: SEQ ID NO: 76 C: SEQ ID NO: 74 C: SEQ ID NO: 75 C: SEQ ID NO: 76 T47-HC GDSVSSNSVA WN R TYYRSKWYN DYAVSVKS ARADGSRGGGYDQ K: SEQ ID NO: 77, res. 6-12  K: SEQ ID NO: 78 K: SEQ ID NO: 79, res. 3-13 I: SEQ ID NO: 77, res. 1-10  I: SEQ ID NO: 78, res. 2-10 I: SEQ ID NO: 79 C: SEQ ID NO: 77 C: SEQ ID NO: 78 C: SEQ ID NO: 79 T47-LC KSS QSILYRSNNKNY LA WAS TRES QQYYTTPQ T K: SEQ ID NO: 80 K: SEQ ID NO: 81 K: SEQ TD NO: 82 I: SEQ ID NO: 80, res. 4-15  I: SEQ ID NO: 81, res. 1-3 I: SEQ ID NO: 82, res. 1-8 C: SEQ ID NO: 80 C: SEQ ID NO: 81 C: SEQ ID NO: 82 m1Q-HC GFTFSSYA MH V ISYDGSNK YYVDSVKG ARSTYGMDV K: SEQ ID NO: 83, res. 6-10  K: SEQ ID NO: 84 K: SEQ ID NO: 85, res. 3-9 I: SEQ ID NO: 83, res. 1-8 I: SEQ ID NO: 84, res. 2-9 I: SEQ ID NO: 85 C: SEQ ID NO: 83 C: SEQ ID NO: 84 C: SEQ ID NO: 85 m1Q-LC RSS QSLVHSDGNTY LN KVS NRDS MQGTHWWT K: SEQ ID NO: 86 K: SEQ ID NO: 87 K: SEQ ID NO: 88 I: SEQ ID NO: 86, res. 4-14  I: SEQ ID NO: 87, res. 1-3 I: SEQ ID NO: 88 C: SEQ ID NO: 86 C: SEQ ID NO: 87 C: SEQ ID NO: 88 2H5-HC GDSVSSKSAA WN R TYYRSKWHN DYAVS ARGQMGALDV K: SEQ ID NO: 89, res. 6-12  K: SEQ ID NO: 90 K: SEQ TD NO: 91, res. 3-10 I: SEQ ID NO: 89, res. 1-10  I: SEQ ID NO: 90, res. 3-10 I: SEQ ID NO: 91 C: SEQ ID NO: 89 C: SEQ ID NO: 90 C: SEQ ID NO: 91 2H5-LC SGS SSNIGSYY VYWY GNN QRPS QSYDSSLSGVI K: SEQ ID NO: 92 K: SEQ ID NO: 93 K: SEQ TD NO: 94 I: SEQ ID NO: 92, res. 4-11  I: SEQ ID NO: 93, res. 1-3 I: SEQ ID NO: 94 C: SEQ ID NO: 92 C: SEQ ID NO: 93 C: SEQ ID NO: 94 m150-HC GFTFSSYA MH V ISYDGSNK YYADSVKG ARLVAGRSAFDI K: SEQ ID NO: 95, res. 6-10  K: SEQ ID NO: 96 K: SEQ ID NO: 97, res. 3-12 I: SEQ ID NO: 95, res. 1-8 I: SEQ ID NO: 96, res. 2-9 I: SEQ ID NO: 97 C: SEQ ID NO: 95 C: SEQ ID NO: 96 C: SEQ ID NO: 97 m150-LC RAS QSVSSN LA GAS TRAT QQYNNWPPIT K: SEQ ID NO: 98 K: SEQ ID NO: 99 K: SEQ ID NO: 100 I: SEQ ID NO: 98, res. 4-9 I: SEQ ID NO: 99, res. 1-3 I: SEQ ID NO: 100 C: SEQ ID NO: 98 C: SEQ ID NO: 99 C: SEQ ID NO: 100

HCDRs of Antibodies Derived from 2H5 VH-Chain Shuffled Libraries

MAbs HCDR1 HCDR2 HCDR3 #4 VH GDSVSSKSVT WN R TYYRSKWFN DYAVS ARAKMGGMDV K: SEQ ID NO: 101, res 6-12  K: SEQ ID NO: 102 K: SEQ ID NO: 103, res 3-10 I: SEQ ID NO: 101, res. 1-10 I: SEQ ID NO: 102, res. 2-10  I: SEQ ID NO: 103 C: SEQ ID NO: 101 C: SEQ ID NO: 102 C: SEQ ID NO: 103 #31 VH GDSVSSNSAA WN R TYYRSKWYN DYAVS TRQSWHGMEV K: SEQ ID NO: 104, res 6-12  K: SEQ ID NO: 105 K: SEQ ID NO: 106, res 3-10 I: SEQ ID NO: 104, res. 1-10 I: SEQ ID NO: 105, res. 2-10  I: SEQ ID NO: 106 C: SEQ ID NO: 104 C: SEQ ID NO: 105 C: SEQ ID NO: 106 #32 VH GDSVSSNSAA WN R TYYRSKWYN DYAVS ARSIATGTDY K: SEQ ID NO: 107, res 6-12  K: SEQ ID NO: 108 K: SEQ ID NO: 109, res 3-10 I: SEQ ID NO: 107, res. 1-10 I: SEQ ID NO: 108, res. 2-10  I: SEQ ID NO: 109 C: SEQ ID NO: 107 C: SEQ ID NO: 108 C: SEQ ID NO: 109 #69 VH GDSVSSSRAT WN R TYYRSKWFN DYAVS ARAKMGGMDV K: SEQ ID NO: 110, res 6-12  K: SEQ ID NO: 111 K: SEQ ID NO: 112, res 3-10 I: SEQ ID NO: 110, res. 1-10 I: SEQ ID NO: 111, res. 2-10  I: SEQ ID NO: 112 C: SEQ ID NO: 110 C: SEQ ID NO: 111 C: SEQ ID NO: 112 A14 VH GDSVSSNSAA WN R TYYRSKWYN DYAVS ARGTRWGMDV K: SEQ ID NO: 113, res 6-12  K: SEQ ID NO: 114 K: SEQ ID NO: 115, res 3-10 I: SEQ ID NO: 113, res. 1-10 I: SEQ ID NO: 114, res. 2-10  I: SEQ ID NO: 115 C: SEQ ID NO: 113 C: SEQ ID NO: 114 C: SEQ ID NO: 115 A21 VH GDSVSSNSAA WN R TYYRSKWYN DYAVS ARAKVYGVDV K: SEQ ID NO: 116, res 6-12  K: SEQ ID NO: 117 K: SEQ ID NO: 118, res 3-10 I: SEQ ID NO: 116, res. 1-10 I: SEQ ID NO: 117, res. 2-10  I: SEQ ID NO: 118 C: SEQ ID NO: 116 C: SEQ ID NO: 117 C: SEQ ID NO: 118 B103 VH GDSVSSKSAT WN R TYYRSRWFN DYAVS ARGNMGAMDV K: SEQ ID NO: 119, res 6-12  K: SEQ ID NO: 120 K: SEQ ID NO: 121, res 3-10 I: SEQ ID NO: 119, res. 1-10 I: SEQ ID NO: 120, res. 2-10  I: SEQ ID NO: 121 C: SEQ ID NO: 119 C: SEQ ID NO: 120 C: SEQ ID NO: 121 B129 VH GDRVSSNRAA WN R TYYRSQWYN DYAVS ARGTAMG -DA K: SEQ ID NO: 122, res 6-12  K: SEQ ID NO: 123 K: SEQ ID NO: 124, res 3-9 I: SEQ ID NO: 122, res. 1-10 I: SEQ ID NO: 123, res. 2-10  I: SEQ ID NO: 124 C: SEQ ID NO: 122 C: SEQ ID NO: 123 C: SEQ ID NO: 124 B139 VH GDSVSSNSAA WN R TYYRSKWYN DYAVS ARQASNGFDI K: SEQ ID NO: 125, res 6-12  K: SEQ ID NO: 126 K: SEQ ID NO: 127, res 3-10 I: SEQ ID NO: 125, res. 1-10 I: SEQ ID NO: 126, res. 2-10  I: SEQ ID NO: 127 C: SEQ ID NO: 125 C: SEQ ID NO: 126 C: SEQ ID NO: 127 B172 VH GDSVSSNSAA WN R TYYRSKWYN DYAVS ARQGTTGFDY K: SEQ ID NO: 128, res 6-12 K: SEQ ID NO: 129 K: SEQ ID NO: 130, res 3-10 I: SEQ ID NO: 128, res. 1-10 I: SEQ ID NO: 129, res. 2-10 I: SEQ ID NO: 130 C: SEQ ID NO: 128 C: SEQ ID NO: 129 C: SEQ ID NO: 130

HCDRs of Antibodies Derived from A14 VL-Chain Shuffled Libraries

MAbs LCDR1 HCDR2 HCDR3 #8 VL SGS SSNIGNYY VSWY DNA KRPS QSYDNSLSGLV K: SEQ ID NO: 131 K: SEQ ID NO: 132 K: SEQ ID NO: 133 I: SEQ ID NO: 131, res. 4-11 I: SEQ ID NO: 132, res. 1-3 I: SEQ ID NO: 133 C: SEQ ID NO: 131 C: SEQ ID NO: 132 C: SEQ ID NO: 133 #20 VL SGT SSNIGSKY VYWY TND QRPS QSYDSSLRAVV K: SEQ ID NO: 134 K: SEQ ID NO: 135 K: SEQ ID NO: 136 I: SEQ ID NO: 134, res. 4-11 I: SEQ ID NO: 135, res. 1-3 I: SEQ ID NO: 136 C: SEQ ID NO: 134 C: SEQ ID NO: 135 C: SEQ ID NO: 136 #20-m1 VL SGT SSNIGSFY VYWY TND QRPS QSYDSSLRAVV K: SEQ ID NO: 137 K: SEQ ID NO: 138 K: SEQ ID NO: 139 I: SEQ ID NO: 137, res. 4-11 I: SEQ ID NO: 138, res. 1-3 I: SEQ ID NO: 139 C: SEQ ID NO: 137 C: SEQ ID NO: 138 C: SEQ ID NO: 139 #20-m2 VL SGT SSNIGSFY VYWY TND QRPS QSYDSSLRAVV K: SEQ ID NO: 140 K: SEQ ID NO: 141 K: SEQ ID NO: 142 I: SEQ ID NO: 140, res. 4-11 I: SEQ ID NO: 141, res. 1-3 I: SEQ ID NO: 142 C: SEQ ID NO: 140 C: SEQ ID NO: 141 C: SEQ ID NO: 142 #20-m3 VL SGT SSNIGSYY VYWY TND QRPS QSYDSSLRAVV K: SEQ ID NO: 143 K: SEQ ID NO: 144 K: SEQ ID NO: 145 I: SEQ ID NO: 143, res. 4-11 I: SEQ ID NO: 144, res. 1-3 I: SEQ ID NO: 145 C: SEQ ID NO: 143 C: SEQ ID NO: 144 C: SEQ ID NO: 145

In embodiments the invention provides an antibody antigen binding domain comprising a heavy chain variable region (Vh) comprising a CDR1, CDR2 and CDR3 combination and a light chain variable region (Vl) comprising a CDR1, CDR2 and CDR3 combination, or comprising a heavy chain variable region (Vh) and/or a light chain variable region (VI), selected from: m36, 71, 76, T47, m1Q, 2H5, m150; and 4, 31, 32, 69, A14, A21, B103, B129, B139, B172; and 8, 20, 20-m1, 20-m2, 20-m3.

In embodiments the antibody antigen binding domain specifically binds aa11-28 or aa19-25 of pre-S1.

The invention also provides antibodies, particularly monoclonal antibodies, and F(ab) or F(ab)2 comprising a subject binding domain.

The invention also provides novel polynucleotides such as cDNAs and expression vectors, encoding a subject antigen binding domain, and cells comprising such polynucleotides, and non-human animals comprising such cells. The polynucleotides may be operably linked to a heterologous transcription regulating sequence for expression, and may be incorporated into such vectors, cells, etc.

The invention provides methods of using the subject domains to treat HBV or HDV infection, or to induce antibody-dependent cell-mediated cytotoxicity (ADCC), comprising administering the domain to a person determined to have HBV or HDV infection, to have been exposed to HBV or HDV, to be at high risk for HBV or HDV exposure or infection, to be in need of Pre-S1 domain antagonism, or to be otherwise in need thereof. The invention further provides the use of subject compositions for the manufacture of a medicament for HBV or HDV infection, optionally in conjunction with a virus replication inhibitor.

The invention includes all combinations of the recited particular embodiments. Further embodiments and the full scope of applicability of the invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. HBV neutralization by 10 antibodies from 2H5 VH-chain shuffled library selections.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

Unless the context indicates otherwise, the term “antibody” is used in the broadest sense and specifically covers antibodies (including full length monoclonal antibodies) and antibody fragments so long as they recognize HBV/HDV Pre-S1 or otherwise inhibit HBV/HDV. An antibody molecule is usually monospecific, but may also be described as idiospecific, heterospecific, or polyspecific. Antibody molecules bind by means of specific binding sites to specific antigenic determinants or epitopes on antigens. “Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′).sub.2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Natural and engineered antibody structures are well known in the art, e.g. Strohl et al., Therapeutic antibody engineering: Current and future advances driving the strongest growth area in the pharmaceutical industry, Woodhead Publishing Series in Biomedicine No. 11, October 2012; Holliger et al. Nature Biotechnol 23, 1126-1136 (2005); Chames et al. Br J Pharmacol. 2009 May; 157(2): 220-233.

Monoclonal antibodies (MAbs) may be obtained by methods known to those skilled in the art. See, for example Kohler et al (1975); U.S. Pat. No. 4,376,110; Ausubel et al (1987-1999); Harlow et al (1988); and Colligan et al (1993). The mAbs of the invention may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

An “isolated polynucleotide” refers to a polynucleotide segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA, which is part of a hybrid gene encoding additional polypeptide sequence.

A “construct” means any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecule has been linked in a functionally operative manner, i.e. operably linked. A recombinant construct will typically comprise the polynucleotides of the invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the invention.

A “vector” refers any recombinant polynucleotide construct that may be used for the purpose of transformation, i.e. the introduction of heterologous DNA into a host cell. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

An “expression vector” as used herein refers to a nucleic acid molecule capable of replication and expressing a gene of interest when transformed, transfected or transduced into a host cell. The expression vectors comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desired, provide amplification within the host. The expression vector further comprises a promoter to drive the expression of the polypeptide within the cells. Suitable expression vectors may be plasmids derived, for example, from pBR322 or various pUC plasmids, which are commercially available. Other expression vectors may be derived from bacteriophage, phagemid, or cosmid expression vectors.

EXAMPLES

Human Monoclonal Antibodies Block Viral Infection of Hepatitis B and D Virus

Here we disclose human monoclonal antibodies that can block HDV and HBV viral infections. These antibodies were identified from a large phage display antibody library, which was established using peripheral blood mononuclear cells from 93 healthy donors. By selection and screening of the antibody library using pre-S1 domain of HBV envelope protein as a target, a panel of human monoclonal antibodies with neutralizing activities against HBV and HDV infections were identified. Among them, 2H5, showed best neutralizing activities against HBV and HDV infections. The co-crystal structure of 2H5 in complex with its target (8 amino acids of the Pre-S1 domain) was solved. By optimizing 2H5 by chain shuffling approach we developed even more potent neutralizing antibodies. These antibodies recognize similar epitope as 2H5 and the epitope is highly conserved among different genotypes of HBV. An exemplary antibody, A14 was tested in mice bearing humanized NTCP and provided complete protection of mice from HDV infection, and animal studies confirmed protection against HBV infection.

Antigen Target: Pre-S1 Peptides.

As antigen for selection we used two peptides derived from the pre-S1 domain of HBV. They were synthesized by Scilight-peptide (Beijing, China) at purity greater than 95%. NC36b: a peptide comprising of residues 4-38 of the pre-S1 domain of HBV L protein with a biotin modification at its C-terminus. m47b: a myristoylated lipopeptide comprising of amino acids 2-48 of pre-S1 domain with a biotin modification at the C-terminus and a myristoylation modification at the N-terminus.

Pre-S1 GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVG (SEQ ID NO: 146) (2-48) m47b Myr-G.......................................... K-Biotin NC36b N...............................K-Biotin

Human Monoclonal Antibodies Against Pre-S1 Peptides were Generated Based on Phage Display Antibody Technology with Modifications [1, 2].

Phage Display Antibody Library.

A human non-immune scFv (Single-chain variable fragment) antibody library was constructed from peripheral blood mononuclear cells (PBMCs) of 93 healthy donors. The library has a size of a total of 1.1×10¹⁰ members.

Selection and Screening of Phage Antibody Library.

Phage particles expressing scFv on their surface (phage-scFv) were prepared from the library and used for selection of scFvs against the synthesized NC36b and m47b. The peptides were captured on streptavidin-conjugated magnetic M-280 Dynabeads® (Life Technologies) and then incubated with 5×10¹² phage particles prepared from the library, respectively. For each peptide, two rounds of selection were performed. For each round of selection, in order to obtain high affinity antibodies, the amount of peptides captured onto the magnetic beads was optimized and extensive washing steps were applied. In addition, to recover high affinity binders from the magnetic beads and increase the diversity of phage-scFvs recovered, two elution methods including peptide competition elution and conventional basic triethanolamine solution were used. Subsequently, a total of about 2000 single clones were picked and rescued to produce phage-scFvs in the bacterial culture supernatant, and screened for specific binding to m47b and/or NC36b by enzyme-linked immunosorbent assay (ELISA). Clones that bound to m47b and/or NC36b with values of optical density at 450 nm>1.0 were scored as positive, whereas negative clones gave values of <0.1. For m47b and/or NC36b specific binding clones, the genes of variable regions of heavy (VH) and light (VL) chain were sequenced and their corresponding amino acid sequences were aligned to eliminate repeated clones and identify antibodies with different sequence for further characterization. A total of 109 clones with unique sequence were identified.

Further Characterization of the Antibodies with Unique Antibody Sequences to Identify the Best Antibody Candidate.

The antibody clones with unique sequence were either produced as purified phage-scFv particles or converted to scFv-Fc minibodies or full-length human IgG1s, and then tested for their binding activities by ELISA, and HBV and HDV neutralization activities in cell cultures. By these assays, antibodies were ranked based on their binding activity and neutralization activity. The top antibody with the highest neutralization activity was chosen for further development.

Preparation of Purified Phage-scFvs for ELISA or Neutralization Assay.

The phage-scFvs in the supernatant of 10-30 mL bacterial culture were precipitated by PEG/NaCL and then quantified by a spectrometer. Activities of different phage-scFvs for antigen binding or neutralizing viral infection were evaluated based on the dose-response of serial diluted phage-Abs that was normalized to the same concentration.

Preparation of scFv-Fc Minibodies.

ScFv encoding gene from the phage-scFv expressing vector was subcloned into an expression vector containing human IgG1 Fc fragment at C-terminus of the scFv. To produce scFv-Fc, 293F (Life Technologies) or 293T cells (ATCC) were transiently transfected with the scFv-Fc expression plasmid, 72 hours after transfection, the cell culture supernatant were harvested and scFv-Fc was purified by Protein A affinity chromatography (Protein A Sepharose CL-4B, GE Healthcare).

Preparation of Full-Length IgG1 Antibody.

The VH and VL coding sequence of a scFv were separately subcloned into antibody heavy chain (HC) expression vector and light chain (LC) expression vector. To make IgG1 antibody, 293F or 293T cells were transiently co-transfected with the two expression plasmids (HC+LC plasmids) at a 1:1 ratio. 72 hours after transfection, the cell culture supernatant were harvested for purification of IgG1 by Protein A affinity chromatography.

ELISA Assay.

5 μg/mL of streptavidin (Sigma) in phosphate buffered saline (PBS) was coated in U-bottom 96-well plate (Nunc, MaxiSorp™), 100 μL per well, at 4° C. overnight or 37° C. for 1 hour. 2 μg/mL (370 nM) of m47b or NC36b peptides at 100 μL per well were then captured onto the plates by incubation at 30′C for 0.5-1 hour. For phage-scFv based ELISA, serial diluted phage-scFvs in PBS containing 2% nonfat milk were added to each well at 100 μL per well. Specific bound phage-scFvs were detected by adding HRP-conjugated mouse anti-M13 antibody (GE Healthcare) and incubated for 30 mins at 30° C. In between each incubation step, the ELISA plate was washed for 6 times with PBST solution (0.05% Tween20 containing PBS) at 200 μL per well. Followed by HRP-conjugated antibody incubation, the ELISA signal was developed by incubating with TMB substrate (Sigma) for 5-10 mins at 30° C. and then stop the reaction with 2M H₂SO₄ at 25 μL per well. The absorbance at 450 nm was read by a microplate reader (Bio-Rad). For scFv-Fc or IgG1 based ELISA, the method was basically the same as described above for phage-scFvs except the hound antibodies were detected by HRP-conjugated mouse anti-human IgG Fc antibody (Sigma).

Preparation of HBV and HDV Viruses.

HBV and HDV were produced as previously described [3]. HDV. Briefly, a plasmid containing a head to tail trimer of 1.0×HDV cDNA of a genotype I virus (Genebank accession number: AF425644.1) under the control of a CMV promoter was constructed with de novo synthesized HDV cDNA for the production of HDV RNPs. A pUC18 plasmid containing nucleotide 2431˜4990 of HBV (Genotype D, Genebank accession number: U95551.1), was used for expressing HBV envelope proteins under the control of endogenous HBV promoter. HDV virions were produced by transfection of the plasmids in Huh-7 as previously described by Sureau et al [4]. The transfected cell culture supernatant was harvested and directly used for HDV neutralization assay. HBV. HBV genotype B, C and D viruses were produced by transfection of Huh-7 cells with a plasmid containing 1.05 copies of HBV genome under the control of a CMV promoter. Genotype B or C HBV viruses were also from plasma of HBV patients.

HBV and HDV Neutralization Assays.

The neutralization assays were performed as previously described [3, 5] with minor modifications. HepG2-hNTCP cells (a HepG2 cell line stably expressing HBV and HDV receptor hNTCP (human sodium taurocholate cotransporting polypeptide)) were used in these assays. HepG2-hNTCP cells were cultured in PMM medium [3] for 12-24 hours in a 48-well plate before viral infection. About 500 multiplicities of genome equivalents (mge) of HDV or 200 mge of HBV mixed with different forms of antibodies: phage-scFvs, scFv-Fc or IgG1 were inoculated with HepG2-hNTCP cells in the presence of 5% PEG8000 and incubated for 16 hours. Cells were then washed with medium for three times and maintained in PMM. Cell culture medium was changed with fresh PMM medium every 2-3 days. For HDV infection, at 7 days post infection (dpi), HDV infected cells were fixed with 100% methanol at room temperature for 10 min, intracellular delta antigen was stained with 5 μg/mL of FITC conjugated 4G5 (a mouse anti-HDV Delta antigen monoclonal antibody) and nuclear were stained with DAPI. Images were collected by a Fluorescence Microscope (Nikon). The neutralization activity against HDV was determined based on the stained Delta antigen amount and strength. For HBV infection, at dpi 3, 5 and 7, the culture supernatant were collected and tested for HBV secreted viral antigen HBsAg and/or HBeAg with commercial ELISA kits (Wantai, Beijing, China). The levels of HBeAg and/or HBsAg were used to evaluate HBV neutralization activity of the antibodies.

Through the above described ELISA and HBV neutralization assays we identified some top antibodies, which showed specific binding with NC36b as well as m47b and 47b (a peptide similar to m47b but without the myristoylation and showed neutralization activities in HBV.

Among these top antibodies, m36, 2H5 and m1Q were the top three antibodies showing best HBV (genotype D) neutralization activity. m36 was excluded from further testing as it showed reduced expression when converted into full-length IgG1. 2H5 and m1Q were further compared for HDV neutralization activity, 2H5 showed better activity in neutralizing HDV infection. Based on the high binding activity with the peptide and potent neutralizing activity against HBV and HDV, 2H5 was chosen for further development. In addition, 2H5 showed greater HBV and HDV neutralization activity than a previously published pre-S1 peptide antibody KR127 [6-8]. In HBV infection assay, 2H5-IgG1 is 11-fold more potent than KR127 as indicated by the IC₅₀ (the antibody concentration resulting 50% inhibition of HBV infection); 2H5 also showed greater inhibitory effect on HDV infection assay.

Mapping the Binding Epitope of 2H5 Antibody.

To map the epitope of 2H5 on pre-S1 region, we synthesized short peptides covering different regions of the pre-S1 domain and tested their ability to compete for the binding of 2H5 to m47b by competition ELISA assay. The shortest peptide that can compete for the binding is the LN16 peptide (corresponding to the NT amino acid (aa) 11-28 of the pre-S1 domain of HBV L protein (Genotype D), indicating the binding epitope of 2H5 is located within this region. LD15 and LA15 peptides also showed some degree of competition activity but at lower level than LN16. The common amino acids shared by the three peptides, LN16, LD15 and LA15, are aa19-25 of pre-S1. We therefore tested LN16 peptides each carrying a single alanine mutation at position 19, 20, 22 and 23, LN16-L19A, -D20A, -P21A, -F23A, for their competition activity, the result showed that all of them had reduced competition activity (LN16-L19A) or completely lost this activity (LN16-D20A, -P21A, -F23A), indicating these amino acids are critically important for pre-S1 binding to 2H5.

The 2H5 Epitope is Highly Conserved Among the Majority of HBV Genotypes.

Sequence alignment of pre-S1 peptides of eight HBV genotypes showed that the epitope is highly conserved among them. The major variable amino acid is at position 24: glycine in genotype A and C, a lysine or arginine in genotype D and other genotypes. To test if this amino acid change will affect 2H5 binding to pre-S1 peptide, the NC36b peptide containing an arginine at position 24 was synthesized and test for binding with 2H5 by ELISA. The result showed that this amino acid change had only minimal effect on the binding. This is consistent with the HBV and HDV viral neutralization result that 2H5 neutralized HBV of genotype D and HDV carrying HBV genotype D envelopes.

Structural Characterization of the 2H5 scFv and Pre-S1 Peptide Complex.

We also determined the crystal structure of 2H5 (as the scFv fragment fused with a His₆ tag at its N-terminal) in complex with a pre-S1 peptide, 59C. The amino acid sequence of 59C corresponds to aa-10˜48 of pre-S1 of genotype C: GGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQV (SEQ ID NO:147). 2H5-scFv and 59C were co-expressed in E. coli. The complex was purified as a complex by Immobilized Metal Ion Affinity Chromatography (IMAC) using Ni-NTA agarose beads (QIAGEN) followed by Size Exclusion Chromatography-HPLC (SEC-HPLC) with Superdex S200 10/300 column (GE Healthcare). The purified 2H5-scFv/59C complex was then concentrated and crystallized at 20° C. using the hanging-drop vapor-diffusion method by mixing 1 μL of protein (29 mg/mL in 10 mM VI Tris-HCl pH 8.0 and 100 mM NaCl) and 1 μL of reservoir solution containing 2.8 M sodium acetate, pH 7.0. Needle-shaped crystals appeared after 10 days. The X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility beamline BL17U and processed by HKL2000 [9]. The structure was determined at 2.7 A° resolution by molecular replacement in Phaser [10, 11] using VH and VL derived from the structure of Herceptin-Fab complex (PDB 3H0T) [12] as starting model. Initial model from molecular replacement was further refined in Phenix [13] and manually rebuilt with Coot [14]. The final model includes 220 residues of 2H5 scFv, residues 20-27 of the 59C peptide. RAMPAGE analysis shows that 96.71% of residues are in the favored region and 3.29% of residues are in the allowed region [15]. The structure revealed that both VH and VL of 2H5 scFv participate in the interaction with the peptide. The eight amino acids of the peptide included in the structure are D₂₀-P₂₁A₂₂F₂₃G24N₂₅A₂₆S₂₇. Among them, D₂₀, P₂₁, A₂₂, F₂₃, A₂₆ and S₂₇ make interactions with 2H5. Three amino acids, D₂₀, P₂₁ and F₂₃ make critical interactions for 2H5 binding.

Improvement of 2H5 Affinity and Neutralization Activity by VH-Chain Shuffling.

Identification of Four Top Antibodies from VH-Chain Shuffled Library of 2H5.

We next used chain shuffling to improve 2H5's binding affinity and neutralization activity, in which one of the two chains (VH and VL) is fixed and combined with a repertoire of the other chain to yield a secondary library that can be selected for superior activity. First, we did VH chain shuffling, in which VL of 2H5 was fixed and paired with a library of VH chains. Two VH-Lib/2H5VL phage display libraries were constructed. One library size is ˜2×10⁸, the other one is about 9×10⁸. By using peptides captured on streptavidin-conjugated magnetic M-280 Dynabeads® (Life Technologies) as target, the two VH-Lib/2H5VL libraries were separately selected for one round each. At the end of the one round of selection from both libraries, total 576 individual clones were randomly picked and screened for binding with m47b by ELISA. Positive clones in ELISA were selected and sequenced. 10 clones with unique VH sequences (Table 1) and showed equal or stronger binding activity to m47_(b) in phage antibody form than 2H5 were identified. These 10 clones were then converted into full-length human IgG1 and validated for binding to m47b by ELISA, neutralizing HBV (genotype D) (FIG. 1) and HDV by in vitro neutralization assays. Four top antibodies, #31, #32, A14 and A21 were selected based on their overall activities in binding to m47b, neutralizing HBV and HDV.

TABLE 1  VH sequence alignment of 10 antibodies from 2H5 VH-chain shuffled library selections. QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWY #4 VH ......................G........K.VT.....E..TG..............F #31 VH ............................................................ #32 VH ............................................................ #69 VH ...........M...................SR.T.....E..TG..............F 2H5 VH ......................G........K...........................H A14 VH ............................................................ A21 VH ............................................................ B103 VH ......................G........K..T...V...A..............R.F B129 VH ...........L...............R....R.....V..................Q.. 3172 VH ............................................................ B139 VH ..................T...V.....................................  NDYAVSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARGKMGGMDVWGQGTTVTVSS (SEQ ID NO: 148) #4 VH ............V....................RG......A.................. #31 VH .......................................T.QSWH..E............ #32 VH ..............S...........K..............SIAT.T.Y.....L..... #69 VH ............V....................RG......A.................. 2H5 VH ..........................................Q..AL............. A14 VH ..........................................TRW.........L..... A21 VH .........................................A.VY.V............. B103 VH ............VK...............S.....I......N..A.............. B129 VH ..........V..S..A....V....................TAM.-.A.....L..... B172 VH .........................................QGTT.F.Y........... 8139 VH ......L..................................QASN.F.I.....M.....

FIG. 1. shows HBV neutralization by 10 antibodies from 2H5 VH-chain shuffled library selections. HepG2-hNTCP cells were infected by incubation with HBV (genotype D) in the presence of antibodies at different concentrations for 16 hours. Antibody and viruses were washed away afterwards and continued to culture for 7 days, cell culture medium was changed every 2 days. The secreted HBeAg was detected by ELISA at 7 days post infection. Based on the reduction of HBeAg level, the HBV neutralization activity was calculated and expressed as the percentage changes for infected cells in the presence of antibodies relative to the control (cells infected in the presence of a control antibody).

Epitope Mapping of the Four Top Antibodies from 2H5 VH-Chain Shuffled Libraries.

As described above, we used peptide competition ELISA method to map the binding epitope of the four top antibodies identified from 2H5 VH-chain shuffled libraries. The LN16 peptide (corresponding to the NT amino acid (aa) 11-28 of pre-S1 domain), and LN16 peptide mutants, LN16-L19A, -D20A, -P21A, -F23A were used to compete for binding of these antibodies to m47b peptide. Our data revealed that all of them had similar peptide competition pattern as 2H5, amino acids, L19, D20, P21 and F23 are important for these antibodies' binding. The D20 and F23 are most important for all antibodies, whereas L19 and P21 played slightly variable role for different antibodies.

Further Characterize the Four Top Antibodies from 2H5 VH-Chain Shuffled Libraries.

These antibodies have more than 15-20 fold improved HBV (genotype D) neutralization activity as compared to the parental 2H5 antibody. The 1050 for these antibodies are around ˜10-40 pM. A representative antibody out of these 4 antibodies, A14, was further compared to Hepatitis B Immune Globulin in neutralizing HBV (genotype D) infection. HBIG is prepared from the plasma of donors who have high antibody levels of the hepatitis B surface antigen (HBsAg) and used as a post exposure prophylaxis for people at risk to develop hepatitis B in clinic. A14 showed more than 1000-fold greater neutralization activity than HBIG. Furthermore, A14 showed broadly neutralization activity against other two HBV genotypes, B and C. The IC50 for genotype B, C and D are 80 pM, 30 pM and 10 pM, respectively. A14 was also examined for neutralizing six HBV genotype C viruses from plasma of HBV infected patients. Again A14 was at least several hundreds to 1000-fold more potent than HBIG in neutralizing these viruses.

A14 is the one with the highest Fab melting temperatures (Tm) of 80.2° C., reflecting the best thermostability of its variable domains. A14 is stabilized by approximately 2° C. comparing to the original 2H5, whereas other three nAbs all have slightly reduced thermostability. The thermostability was measured using differential scanning calorimetry (DSC).

Using primary human hepatocytes (PHH), we also demonstrated the potent neutralization activity of A14 against two HBV clinical strains from HBV patient plasma samples. One virus is genotype B; the other virus is a genotype C virus. HBsAg or HBeAg secreted to cell culture supernatants was examined every two days over the entire infection course using commercial kits (Autobio Diagnostics Co., Ltd.).

A14 competed with pre-S1 for binding to NTCP expressed on cells. A14 effectively competed with pre-S1 (FITC labeled pre-S1 peptide: m59) for binding to NTCP expressed on HepG2 cells in a dose-dependent manner.

A14 has no cross reactivity with 12 different cell lines representing 6 different tissues. This was analyzed by Western blotting and immunostaining assays.

A14 has antibody mediated cytotoxicity (ADCC) activity against cells carrying its epitope on cell surface and HBV producing cells as well as infected cells. In the ADCC assay, the epitope of A14 was stably expressed on CHO cell surface, HBV producing DE19 cells, and infected HepG2-hNTCP cells were used as target cells. A human NK cell line (NK92-MI expressing CD16 (V158 allele) and FcRgamma chain was used as effector cells. The effector cells and target cells (E/T) were co-cultured at a ratio of 6:1 for 6 hours in the presence of A14 or its Fc mutant. The cell killing was determined by using LDH release assay kit form Promega. The ADCC assay showed that A14 exhibited strong specific killing of CHO cells expressing the epitope, HBV producing cells, and HBV-infected HepG2-hNTCP cells but not the control cells lacking of the epitope expression, non-HBV producing cells and non-HBV infected cells. Meanwhile, the A14's Fc mutant (D265A/N297A) that lacks the ADCC activity but retains the same binding activity had no ADCC activity.

ADCC activity is common to antibodies having the same or similar epitope as A14, including 2H5, and its VH chain shuffled derived ones: 4, 31, 32, 69, A14, A21, B103, B129, B139, B172, and the VL chain shuffled clones #8, 20, 20-m1, 20-m2, 20-m3, and antibodies having distinct epitopes, such as m36, 71, 76, T47, m150, m1Q can also present ADCC activity; for example, m1Q, also showed ADCC activity, its epitope is approximate to the C-terminal of A14's epitope on preS1.

A14 Protected Mice from HDV Infection.

We previously revealed that the molecular determinant restricting mouse NTCP (mNTCP) to support viral entry of HBV and HDV is located within the residues 84-87 of mNTCP. When residues 84-87 were replaced by the human NTCP counterparts, it can effectively support viral infections in cell cultures [16]. Based on this, we have established a mouse model (background of FVB strain) that can support HDV infection by replacing mNTCP's residues at 84-87 with the corresponding residues of hNTCP using a genome editing method, TALEN [17, 18]. Using this mouse model, we tested if A14 can protect mice from HDV infection. FVB mice (age of 9 days after birth) with aa84-87 of mNTCP modified homozygotes were administered A14 mAb at 10 mg/kg of body weight. At 1 hour after mAb administration, mice were challenged with HDV viruses. At day 6 after HDV challenge, mice were sacrificed and liver tissues were harvested in liquid nitrogen immediately after collection. Mouse liver samples were then homogenized and lysed by Trizol® reagent to extract the total RNA. The RNA samples were reverse transcribed into cDNA with Prime Script RT-PCR Kit (Takara). To quantify HDV total RNA (genome equivalent) and edited NTCP RNA copies, the cDNA obtained from 20 ng RNA was used as template for real time PCR assay. Real time PCR was performed on an ABI Fast 7500 real time system instrument (Applied Biosystems, USA). The edited NTCP and HDV viral genome equivalent copies were calculated with a standard curve and the cellular GAPDH RNA was used as an internal control. A14 mAb completely blocked HDV infection, whereas HDV infection reached 1-10×10⁶ copies/20 ng liver RNA in the control group. Mice in both groups had comparable NTCP mRNA copies in the liver tissue.

A14 Protected Mice from HBV Infection in a Prophylaxis Mouse Model and Inhibited HBV Infection in a Treatment Mouse Model.

A mouse HBV infection model has been established using FRG (Fah−/−Rag2−/−/IL2rg−/−) triple knock-out mice transplanted with human hepatocytes [19, 20]. The FRG mice allows transplanted human hepatocytes replicating in mouse liver to form a chimeric liver with up to 98% human hepatocytes, as such the liver humanized FRG mice (FRGC) are highly susceptible to HBV infection. To test the prophylactic effect of A14, 10 FRGC mice were divided into two groups, five mice each. A14 prophylaxis group mice were injected with A14 at 15 mg/kg dosage by a single IP administration one day prior to HBV virus challenge, while mice in the control group were injected with same volume of PBS. On day 0, all mice were injected with 10e9 GE (genome equivalent) HBV each via tail vein. To test the therapeutic effect of A14, FRGC mice were challenged with 10e9 GE/mice of HBV via tail vein on day 0, on day 5 post-infection, the mice were treated with entecavir (ETV) control or A14 or HBIG. ETV was orally given at 0.1 mg/kg daily; A14 or HBIG were administrated every three days by I.P. injection at 20 mg/kg and 72 mg/kg (40 IU/kg), respectively. For both prophylaxis and treatment model, blood samples were collected every 3 days from all mice for measuring HBsAg and HBV DNA titer in serum. The mice were scarified at the end of the experiment, dpi35 and the liver tissues were preserved for immunohistochemical staining (THC) of HBsAg and HBcAg. A14 showed 100% protection of FRGC mice from HBV infection in the prophylaxis model; it also showed significant inhibition of HBV infection in the treatment model.

Taken together, the results clearly demonstrated that A14 mAb is a potent HDV and HBV entry inhibitor in animal model. A14 mAb can be used to replace HBIG for prevention of HDV and HBV infection. On the other hand, A14 treatment of an established HBV infection in mice significantly inhibited HBV infection, moreover A14 showed specific ADCC activity against HBV-infected cells but not the non-HBV infected cells. These results indicate that A14 mAb may be combined with ETV to treat patient who are chronically infected by HBV. As A14 blocks new viral entry into host cells and has ADCC activity against infected cells, whereas ETV inhibits viral replication, combination of A14 with a viral replication inhibitor such as ETV, lamivudine, adefovir, tenofovir, telbivudine or other nucleoside and nucleotide analogues (NUCs) provide new therapeutic and prophylactic options for patients and can achieve better viremia control and HBsAg reduction.

Improvement of A14 Affinity and Neutralization Activity by VL-Chain Shuffling.

To further improve A14 activity, we made an A14-VL chain shuffled phage display library, in which VH of A14 was fixed and paired with a library of VL chains. The final library (A14VH/VLlib) constructed had a size of ˜3×10⁸. By using m47b peptide captured on streptavidin-conjugated magnetic M-280 Dynabeads® (Life Technologies) as target, the A14VH/VLlib library was selected for two rounds. 196 clones were screened for binding with m47b by ELISA. All clones were positive but 24 clones with highest OD450 reading were picked for sequencing. Two clones, #8 and #20, with different VL chain sequences than A14's VL were identified. These two antibodies were converted into full-length human IgG1 and tested for binding to m47b by ELISA. They both showed stronger binding activity to m47b than A14. In the HBV neutralization assay of HBV (genotype D), #8 showed 5-fold improvement in neutralizing HBV infection, whereas #20 showed similar activity as A14. Further mutagenesis of the VL of #20 (#20-m1, -m2, -m3) improved its neutralization activity by ˜3-5-fold than A14, reached to the similar level as #8. The elevated HDV neutralization activities of these #20 mutants compared to A14 were demonstrated. Thus these A14-derived antibodies with further improved activities can be used similarly as A14 as described above, either alone or in combination with a viral replication inhibitor.

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m36 m36 VH DNA: (SEQ ID NO: 01) CAAGTTCCTTTATGTGCTGTCTCATCATTTTGGCAAGAATTCGCCACCATGAAACATCTGTGGT TCTTCCTTCTCCTGGTGGCAGCGGCCCAGCCGGCCATGGCCCAGATGCAGCTGGTGCAGTCTGG GGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTT GATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAG AGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTG TATTACTGTGCAAAAACGTCCTACGGGGGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCA CCGTCTCCTCA  m36 VL DNA: (SEQ ID NO: 02) CAGCCTGTGCTGACTCAATCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAAACACTTCCAACATCGGAAGTTATTATGCATACTGGTATCAGCAACTCCCAGGAAC GOCCCCCAAACTCCTCATCTATGATAATAATCAGCGGCCCTCOGGGATCCCTOCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCAG ATTATTACTGTGCAACATGGGATGACAGCCTGAATGGTCCGGTGTTCGGCGGAGGGACCAAGGT CACCGTCCTA  m36 VH Amino acid: (SEQ ID NO: 03) QMQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSV KGRFTTSRDNAKNSLYLQMNSLRAEDTALYYCAKTSYGGAFDIWGQGTMVTVSS  m36 VL Amino acid: (SEQ ID NO: 04) QPVLTQSPSASGTPGQRVTTSCSGNTSNIGSYYAYWYQQLPGTAPKLLIYDNNQRPSGTPARFS GSKSGTSASLAISGLQSEDEADYYCATWDDSLNGPVFGGGTKVTVL  71: 71 VH DNA: (SEQ ID NO: 05) CAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCT GCAAGGCTTCTGGATACACCTTCACCGGCTACTATATACATTGGGTGCGACAGGCCCCTGGACA AGGGCTTGAGTGGATGGGACGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTT CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGGACGGCCTACATGGAACTGAGTACAC TGACATCTGACGACACGGCCOTTTATTACTGTGCGAGAGAAGGAAGGGGCGGCATGGACGTCTG GOGCCAAGGGACCACGOTCACCGTCTCCTCA  71 VL DNA: (SEQ ID NO: 06) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCT CCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCA GAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTOGGTTCTAATCGGGCCTCCGGGOTCCCT CACAGGTTCACTCCCACTCCATCACGCACACATTTTACACTGAAAATCACCACAGTGGAGCCTC ACCATGTTCCCATTTATTACTOCATGCAACCTCTACAACCTCCCATCACCTTCGGCCAGGGGAC ACGACTGGAGATTAAA  71 VH Amino acid: (SEQ ID NO: 07) QVQLVESGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGRINPNSGGTNYAQKF QGRVTMTRDTSIRTAYMELSTLTSDDTAVYYCAREGRGGMDVWGQGTTVTVSS  71 VL Amino acid: (SEQ ID NO: 08) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP DRFSGSGSGTDFTLKISRVEAEDVGTYYCMQGLQPPITFGQGTRLEIK  76: 76 VH DNA: (SEQ ID NO: 09) GAGGTGCAGCTGTTGGAGACCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTG AACCGCCGATTCACCATCTCCACAGACAATTCCAAGAACACCCTCTATCTCCAAATGAACACCC TCAGACCTGACCACACCCCTCTCTATTACTGTGGGAGTGGTGCTTTTGATATCTGCGCCCAACC GACAATGGTCACCGTCTCTTCA  76 VL DNA: (SEQ ID NO: 10) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCT CCTGCAGGTCTAGTCACAGCCTCGTATACAGTGATGGAAACACCTACTTGAGTTGGTTTCACCA GAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAATCGGGACTTTGGGGTCCCA GACAGATTCAGCGGCAGTGGGTCAGGCACTGACTTCACACTGAAGATCAGCAGGGTGGAGGCTG AGGATGTTGGAGTTTATTACTGCATGCAAGGTACACACTGGCCTGGGACGTTCGGCCAGGGGAC CAAACTGGATATCAAA  76 VH Amino acid: (SEQ ID NO: 11) EVQLLETGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSV KGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCASGAFDIWGQGTMVTVSS 76 VL Amino acid: (SEQ ID NO: 12) DVVMTQSPLSLPVTLGQPASISCRSSHSLVYSDGNTYLSWFHQRPGQSPRRLIYKVSNRDFGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPGTFGQGTKLDIK  T47: T47 VH DNA: (SEQ ID NO: 13) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCTCCT GTGCCATCTCCGGGGACAGTGTCTCCAGCAACAGTGTTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTCTCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAGCTCTGTGACTCCCGAGGACACGGCTGTATATTACTGTGCAAGAGCCGATGGTTCGCGAGG GGGAGGGTATGACCAGTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA  T47 VL DNA: (SEQ ID NO: 14) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCA AATGCAAGTCCAGTCAGTCTATTTTATACAGGTCCAACAATAAGAACTACTTAGCTTGGTACCA ACACAAACCAGGACAGCCTCCTAAGCTGCTCATTTCCTGGGCATCTACCCGGGAATCCGGGGTC CCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGG CTGAAGATGTGGCGGTTTATTACTGTCAGCAATATTATACTACTCCTCAGACTTTTGGCCAGGG GACCAAGGTGGAGATCAAA  147 V14 Amino acid: (SEQ ID NO: 15) QVQLQQSGPGLVKPSQTLSLSCAISGDSVSSNSVAWNWIRQSPSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNPDTSKNQFSLQLSSVTPEDTAVYYCARADGSRGGGYDQWGQGTLVTVSS   T47 VL Amino acid: (SEQ ID NO: 16) DIVMTQSDDSLAVSLGERATTKCKSSQSILYRSNNKNYLAWYQHKPGQDDKLLISWASTRESGV DDRFSCSGSCTDFTLTTNSLQAEDVAVYYCQQYYTTDQTFCQCTKVEIK  m1Q m1Q VH DNA (SEQ ID NO: 17) CAGGTCCAGTTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGCAGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGTAGACTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGATCTACATACGGTATGGACGTCTGGGG CCAAGGGACCACGGTCACCGTCTCCTCA  m1Q-VL DNA (SEQ ID NO: 18) GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGTCGGCCTCCATCT CCTGCACGTCTACTCAAAGCCTCGTACACAGTCATGGAAACACCTACTTGAATTGOTTTCACCA GAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAATCGGGACTCCGGGGTCCCA GACAGATTCAGCGGCAGTGGGTCAGACACTGATTTCACACTGGAAATCAGCAGGGTGGAGGCCG ACGATCTTGGCATTTATTACTCCATGCAAGGTACACACTGGTCCACCTTCGCCCAAGCCACCAA GCTGGATATCAAA  m10 VH Amino acid: (SEQ ID NO: 19) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEQVAVISYDGSNKYYVDSV KGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCARSTYGMDVWGQGTTVIVSS  m1Q Vk Amino acid: (SEQ ID NO: 20) DVVMTQSPLSLPVTLGQSASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVP DRFSGSGSDTDFTLEISRVEAEDVGTYYCMQGTHWWTFGQGTKLDIK  2H5: 2H5 VH DNA: (SEQ ID NO: 21) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGGCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC TTCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGCATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTTTCCCTGCAGC TGAACTCTGTGACCCCCGAAGACACGGCTGTGTATTATTGTGCGCGCGGCCAGATGGGAGCTTT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  2H5 VL DNA: (SEQ ID NO: 22) CAGTCTGTGTTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAAGCAGCTCCAACATCGGAAGTTATTATGTATACTGGTACCAGCAATTCCCAGGAAC GGCCCCCAAACTCCTCATCTATGGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGTCAGTCCTATGACAGCAGCCTGAGTGGTGTGATATTCGGCGGAGGGACCAAGCT GACCGTCCTA  2H5 VH Amino acid: (SEQ ID NO: 23) QVQLQQSGPGLVKPSQTLSLTCGTSGDSVSSKSAAWNWIRQSPSRGLEWLGRTYYRSKWHNDYA VSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARGQMGALDVWGQGTTVTVSS  2H5 VL Amino acid: (SEQ ID NO: 24) QSVLTQPPSASGTPGQRVTTSCSGSSSNIGSYYVYWYQQFPGTAPKLLIYGNNQRPSGVPDRFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGVIFGGGTKLTVL  m150 m150 VH DNA: (SEQ ID NO: 25) GAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGTTGGTGGCTGGTCGAAGTGCTTTTGA TATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  m150 VK DNA: (SEQ ID NO: 26) GAAATTGTGCTGACTCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGC AGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTT ATTACTGTCAGCAGTATAATAACTGGCCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGAT TAAA  m150 VH Amino acid: (SEQ ID NO: 27) EVQLVQSGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSV KGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCARLVAGRSAFDIWGQGTTVTVSS  m150 NK Amino acid: (SEQ ID NO: 28) EIVLTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGTPARFSG SGSGTEFILTTSSLQSEDFAVYYCQQYNNWPPITEGQGTRLEIK  Antibody Sequences of 10 Antibodies Derived from 2H5 VH-Chain Shuffled Library Selection.

Note, these antibodies have the same VL sequence as 2H5, therefore only VH sequences of these antibodies were listed below.

#4 #4 VH DNA: (SEQ ID NO: 29) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGGCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGTTACTTGGAACTGGATCAGGGAGTCTCC AACGGGAGGCCTTGAGTGGCTGGGCAGGACATACTATAGGTCCAAGTGGTTTAATGATTATGCA GTATCTGTGAAAAGTCGAATAACTGTCAACCCAGACACATCCAAGAACCAGTTTTCCCTGCAGC TAAACTCTGTGACTCCCGAGGACAGGGGTGTCTATTACTGCGCACGCGCCAAGATGGGAGGTAT GGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCTTCA  #4 VH Amino Acid: (SEQ ID NO: 30) QVQLQQSGPGLVKPSQTLSLTCGTSGDSVSSKSVTWNWIRESPIGGLEWLGRTYYRSKWFNDYA VSVKSRITVNPDTSKNQFSLQLNSVTPEDRGVYYCARAKMGGMDVWGQGTTVTVSS  #31 VH DNA: (SEQ ID NO: 31) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TCAACTCTCTGACTCCCCACCACACGCCTCTTTATTACTCTACAACACACACTTGCCACGCTAT CCAAGTCTCCCCCCAACCCACCACCGTCACCCTCTCCTCA  #31 VH Amino acid: (SEQ ID NO: 32) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCTRQSWHGMEVWGQGTTVTVSS  #32 VH DNA: (SEQ ID NO: 33) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACTCAGACACATCGAAGAACCAGTTCTCCCTGCAGC TGAAGTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGGAGTATAGCAACAGGTAC TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  #32 VH Amino acid: (SEQ ID NO: 34) QVQLQQSGPCLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRCLEWLCRTYYRSKWYNDYA VSVKSRITTNSDTSKNQFSLQLKSVTPEDTAVYYCARSIATGTDYWGQGTLVTVSS  #69 VH DNA: (SEQ ID NO: 35) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGATGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGTAGCCGTGCTACTTGGAACTGGATCAGGGAGTCTCC AACGGGAGGCCTTGAGTGGCTGGGCAGGACATACTATAGGTCCAAGTGGTTTAATGATTATGCA GTATCTGTGAAAAGTCGAATAACTGTCAACCCAGACACATCCAAGAACCAGTTTTCCCTGCAGC TAAACTCTGTGACTCCCGAGGACAGGGGTGTCTATTACTGCGCACGCGCCAAGATGGGAGGTAT GGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA  #69 VH Amino acid: (SEQ ID NO: 36) QVQLQQSGPGLMKPSQTLSLTCAISGDSVSSSRATWNWIRESPIGGLEWLGRTYYRSKWFNDYA VSVKSRITVNPDTSKNQFSLQLNSVIPEDRGVYYCARAKMGGMDVWGQGTTVTVSS  A14 VH DNA: (SEQ ID NO: 37) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT CTCCCATCTCCCCCCACACTCTCTCTACCAACAGTCCTCCTTCCAACTCCATCAGCCACTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGAACACGTTGGGGTAT GGACGTCTGGGGCCAAGGGACCCTGGTCACTGTCTCCTCA  A14 VH Amino acid: (SEQ ID NO: 38) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSFSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNFDTSKNQFSLQLNSVTFEDTAVYYCARGTRWGMDVWGQGTLVTVSS  A21 VH DNA: (SEQ ID NO: 39) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGCGAAAGTGTACGGTGT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  A21 VH Amino acid: (SEQ ID NO: 40) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSFSRGLEWLGRTYYRSKWYNDYA VSVKSRITTNFDTSKNQFSLQLNSVTFEDTAVYYCARAKVYGVDVWGQGTTVTVSS B103 VH DNA: (SEQ ID NO: 41) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGGCATCTCCGGGGACAGTGTCTCTAGCAAGAGTGCCACTTGGAACTGGGTCAGGCAGTCCGC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAGGTGGTTTAATGATTATGCA GTGTCTGTGAAAAGTCGAATAACCGTCAAGCCAGACACATCCAAGAACCAGTTTTCCCTGCAAT TAAATTCTGTGAGTCCCGAGGACACGGCTATCTATTACTGTGCACGCGGCAACATGGGAGCTAT GGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTTCA  B103 VH Amino acid: (SEQ ID NO: 42) QVQLQQSGPGLVKPSQTLSLTCGTSGDSVSSKSATWNWVRQSASRGLEWLGRTYYRSRWFNDYA VSVKSRITVKPDTSKNQFSLQLNSVSPEDTAIYYCARGNMGAMDVWGQGTTVTVSS  B129 VH DNA: (SEQ ID NO: 43) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGCTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGGGTCTCTAGCAATAGAGCTGCTTGGAACTGGGTCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCCAGTGGTATAATGATTATGCA GTCTCTGTAAAAAGTCGAGTGACCATCAGCCCAGACGCATCCAAGAACCAAGTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGGTACAGCTATGGGTGA CGCCTGGGGCCAGGGAACCCTGGTCACCGTCTCTTCA  B129 VH Amino acid: (SEQ ID NO: 44) QVQLQQSGPGLLKPSQTLSLTCAISGDRVSSNRAAWNWVRQSPSRGLEWLGRTYYRSQWYNDYA VSVKSRVTTSPDASKNQVSLQLNSVTPEDTAVYYCARGTAMGDAWGQGTLVTVSS  B139 VH DNA: (SEQ ID NO: 45) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCACACTCACCT GTGTCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA CTTTCTCTCAAAACTCCAATAACCATCAACCCAGACACATCCAACAACCACTTCTCCCTCCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGACAAGCCTCCAACGGTTT TGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA  B139 VH Amino acid: (SEQ ID NO: 46) QVQLQQSGPGLVKPSQTLTLTCVISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYA VSLKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARQASNGFDIWGQGTMVTVSS  B172 VH DNA: (SEQ ID NO: 47) CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCT GTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCC ATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACAGGTCCAAGTGGTATAATGATTATGCA GTATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAGC TGAACTCTGTGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGACAGGGGACGACAGGCTT TGACTACTGGGGCCAGGGAACCACGGTCACCGTCTCCTCA  B172 VH Amino acid: (SEQ ID NO: 48) QVQLQQSGPCLVKPSQTLSLTCAISCDSVSSNSAAWNWIRQSPSRCLEWLCRTYYRSKWYNDYA VSVKSRITTNPDTSKNQFSLQLNSVTPEDTAVYYCARQGTTGFDYWGQGTTVTVSS Antibody Sequences of Two Antibodies Derived from A14 VL-Chain Shuffled Library Selection.

Note, these antibodies have the same VH sequence as A14, therefore only VL sequences of these two antibodies were listed below.

#8 VL DNA (SEQ ID NO: 49) CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCT GCTCTGGAAGCAGCTCCAACATTGGGAATTATTATGTGTCCTGGTACCAGCACCTCCCAGGAAC AGCCCCCAAACTCCTCATTTATGACAATGCTAAGCGACCCTCAGGGATTCCTGACCGATTCTCT GGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACTGGGCTCCGGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAATAGCCTTAGTGGTTTGGTGTTCGGCGGAGGGACCAAGCT GACCGTCCTA  #8 VL amino acid: (SEQ ID NO: 50) QSVVTQPPSVSAAPCQKVTTSCSCSSSNTCNYYVSWYQHLPCTAPKLLIYDNAKRPSCIPDRFS GSKSGTSATLGTTGLRAEDEADYYCQSYDNSLSGLVFGGGTKLTVL  #20 VL DNA: (SEQ ID NO: 51) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTAAGTATGTATACTGGTACCAGCGGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGCTGTGGTTTTCGGCGGAGGGACCAAGCT GACCGTCCTA  #20 VL amino acid: (SEQ ID NO: 52) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSKYVYWYQRLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL  #20-m1 VL DNA: (SEQ ID NO: 53) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTTTCTATGTATACTGGTACCAGCGGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT CGCTCCAACTCTGCCACCTCACCCTCCCTCCCCATCACTCCGCTCCAGGCTCAGGATCAGCCTC ATTATTACTCCCACTCCTATCACACCACCCTCCGTCCTCTCCTTTTCCGCCCAGGCACCAACCT GACCGTCCTA  #20-m1 VL amino acid: (SEQ ID NO: 54) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSFYVYWYQRLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL  #20-m2 VL DNA: (SEQ ID NO: 55) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTTTCTATGTATACTGGTACCAGCAGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGCTGTGGTTTTCGGCGGAGGGACCAAGCT GACCGTCCTA  #20-m2 VL amino acid: (SEQ ID NO: 56) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSFYVYWYQQLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL  #20-m3 VL DNA: (SEQ ID NO: 57) CAGTCTGTGTTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTT GTTCTGGAACCAGCTCCAACATCGGAAGTTACTATGTATACTGGTACCAGCAGCTCCCAGGAAC GGCCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGCCCGATTCTCT GGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTG ATTATTACTGCCAGTCCTATGACAGCAGCCTGCGTGCTGTGGTTTTCGGCGGAGGGACCAAGCT GACCGTCCTA  #20-m3 VL amino acid: (SEQ ID NO: 58) QSVLTQPPSASGTPGQRVTTSCSGTSSNIGSYYVYWYQQLPGTAPKLLIYTNDQRPSGVPARFS GSKSGTSASLAITGLQAEDEADYYCQSYDSSLRAVVFGGGTKLTVL  

What is claimed is:
 1. An antibody, or antigen-binding fragment thereof, comprising: a) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 4; b) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 7 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 8; c) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 12; d) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 15 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 16; e) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 19 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 20; f) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 23 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; g) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 27 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 28; h) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 30 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; i) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 32 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; j) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 34 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; k) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 36 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; l) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; m) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 40 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; n) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 42 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; o) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 44 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; p) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 46 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; q) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 48 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 24; r) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 50; s) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 52; t) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 54; u) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 56; or v) a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 38 and a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO:
 58. 2. The antibody, or antigen-binding fragment thereof of, of claim 1, wherein the antibody or antigen-binding fragment thereof further comprises a Fc domain.
 3. The antibody, or antigen-binding fragment thereof of, of claim 1, wherein the antibody or antigen-binding fragment thereof exhibits antibody-dependent cell-mediated cytotoxicity (ADCC) activity in an ADCC assay.
 4. The antibody, or antigen-binding fragment thereof of, of claim 1, wherein the antibody or antigen-binding fragment thereof binds to Pre-S1 of Hepatitis B virus (HBV).
 5. The antibody, or antigen-binding fragment thereof of, of claim 1, wherein the antibody is a monoclonal antibody.
 6. The antibody, or antigen-binding fragment thereof of, of claim 5, wherein the antibody is a human monoclonal antibody.
 7. A method of treating or preventing Hepatitis B virus (HBV) or Hepatitis D virus (HDV) infection in a subject, comprising administering to the subject the antibody or antigen-binding fragment thereof of claim 1, wherein the subject is determined to have HBV or HDV infection or has been exposed to HBV or HDV.
 8. The method of claim 7, wherein the antibody or antigen-binding fragment thereof is administered by injection. 